The Class III Biological Safety Cabinet is a totally enclosed, ventilated cabinet of gas-tight construction and offers the highest degree of personnel and environmental protection from infectious aerosols, as well as protection of research materials from microbiological contaminants. Class III cabinets are most suitable for work with hazardous agents that require Biosafety Level 3 or 4 containment.

All operations in the work area of the cabinet are performed through attached arm length rubber gloves or half-suits. The Class III cabinet is operated under negative pressure. Supply air is HEPA-filtered and the cabinet exhaust air is filtered through two HEPA filters in series, or HEPA filtration followed by incineration, before discharge outside of the facility.

All equipment required by the laboratory activity, such as incubators, refrigerators, and centrifuges, must be an integral part of the cabinet system. The Class III cabinet must be connected to a double-doored autoclaves and/or chemical dunk tank used to sterilize or disinfect all materials exiting the cabinet, and to allow supplies to enter the cabinet. Several Class III cabinets are therefore typically set up as an interconnected system.